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| Functional assessment of mESC derived astrocytes. (A) Time-lapse heat-map of activated astrocytes after stimulation (A) via adenosine triphosphate (ATP) (50 µM). (B) Decrease in percentage of non-responsive cells after ATP stimulation, over the course of 4 weeks. (C) DIV 7-DIV 28 astrocytes treated with suramin exhibit stunted Ca 2+ responses after ATP application (red traces), compared to untreated astrocytes (blue traces), * P < 0.05.
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Astrocytes are multifunctional cells in the CNS, involved in the regulation of neurovascular coupling, the modulation of electrolytes, and the cycling of neurotransmitters at synapses. Induction of astrocytes from stem cells remains a largely underdeveloped area, as current protocols are time consuming, lack granularity in astrocytic subtype genera...
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Context 1
... examined whether the generated astrocytes had functionally mature and responsive purinergic pathways ( Figure 6A). Stimulation of astrocytes via ATP (50 µM) generated slow wave-like Ca 2+ transients, a hallmark of astrocyte function ( Bazargani and Attwell, 2016;Shigetomi et al., 2016). ...
Context 2
... of astrocytes via ATP (50 µM) generated slow wave-like Ca 2+ transients, a hallmark of astrocyte function ( Bazargani and Attwell, 2016;Shigetomi et al., 2016). A timelapse sequence of a 5 minute recording is illustrated in Figure 6A, where calcium concentration in multiple regions of interest (e.g., middle right and middle left) increases (from 30 to 60 s) and returns to initial levels (from 90 to 300 s). Quantification of the percentage of astrocytes displaying transients showed a significant (p < 0.05) decrease in percentage of non-responsive cells from approximately 20% at DIV 7 to approximately 5% at DIV 28 ( Figure 6B). ...
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... timelapse sequence of a 5 minute recording is illustrated in Figure 6A, where calcium concentration in multiple regions of interest (e.g., middle right and middle left) increases (from 30 to 60 s) and returns to initial levels (from 90 to 300 s). Quantification of the percentage of astrocytes displaying transients showed a significant (p < 0.05) decrease in percentage of non-responsive cells from approximately 20% at DIV 7 to approximately 5% at DIV 28 ( Figure 6B). Cellular responses to ATP stimulus were inhibited by the broad-spectrum P2X/2Y antagonist suramin, which blocks metabotropic (P2Y 1 R) and ionotropic (P2X 7 ) purinoceptors ( Bernstein et al., 1998). ...
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... responses to ATP stimulus were inhibited by the broad-spectrum P2X/2Y antagonist suramin, which blocks metabotropic (P2Y 1 R) and ionotropic (P2X 7 ) purinoceptors ( Bernstein et al., 1998). Upon ATP stimulation, we observed a stunted response in suramin treated cells (Figure 6C) across all time-points, providing further validation that prior Ca 2+ responses were indeed purinergic. ...
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Citations
... When the exogenous BRN4 remains expressed, the astrocyte differentiation is severely compromised. Astrocytes can be induced from mESCs by embryoid body (EB) formation followed by retinoic acid treatment with similar efficiency to our SrC switch [66]. Although the current version of the SrC switch using BRN4 does not necessarily improve the astrocyte differentiation system, the SrC switch is easily adaptable for further improvement of astrocyte induction; for example, by manipulating the expression pattern of BRN4, by altering BRN4 functions via mutagenesis, or by using an alternate transcription factor. ...
Background
Viral vectors are attractive gene delivery vehicles because of their broad tropism, high transduction efficiency, and durable expression. With no risk of integration into the host genome, the vectors developed from RNA viruses such as Sendai virus (SeV) are especially promising. However, RNA-based vectors have limited applicability because they lack a convenient method to control transgene expression by an external inducer.
Results
We engineered a Csy4 switch in Sendai virus-based vectors by combining Csy4 endoribonuclease with mutant FKBP12 (DD: destabilizing domain) that becomes stabilized when a small chemical Shield1 is supplied. In this Shield1-responsive Csy4 (SrC) switch, Shield1 increases Csy4 fused with DD (DD-Csy4), which then cleaves and downregulates the transgene mRNA containing the Csy4 recognition sequence (Csy4RS). Moreover, when Csy4RS is inserted in the viral L gene, the SrC switch suppresses replication and transcription of the SeV vector in infected cells in a Shield1-dependent manner, thus enabling complete elimination of the vector from the cells. By temporally controlling BRN4 expression, a BRN4-expressing SeV vector equipped with the SrC switch achieves efficient, stepwise differentiation of embryonic stem cells into neural stem cells, and then into astrocytes.
Conclusion
SeV-based vectors with the SrC switch should find wide applications in stem cell research, regenerative medicine, and gene therapy, especially when precise control of reprogramming factor expression is desirable.
... 39 In this study, we committed mouse embryonic stem cells to neural lineages, by fine tuning a mass suspension protocol by Wichterle and Peljto 40 to generate embryoid bodies (EBs) within 5 days. This fast and efficient adaptation has produced both astrocytes 41 and neurons. 42,43 Hence, the objectives of this study were to examine the subcellular localisation and colocalisation of endogenously expressed ERα, GPER-1 and ERα-36 in neurons differentiated from pluripotent mouse stem cells. ...
Oestrogen receptors (ER) transduce the effects of the endogenous ligand, 17β‐estradiol in cells to regulate a number of important processes such as reproduction, neuroprotection, learning and memory and anxiety. The ERα or ERβ are classical intracellular nuclear hormone receptors while some of their variants or novel proteins such as the GPCR, GPER1/GPR30 are reported to localise in intracellular as well as plasma membrane locations. Though the brain is an important target for oestrogen with oestrogen receptors expressed differentially in various nuclei, subcellular organisation and crossttalk between these receptors is under‐explored. Using an adapted protocol that is rapid, we first generated neurons from mouse embryonic stem cells. Our immunocytochemistry approach shows that the full length ERα (ERα‐66) and for the first time, that an ERα variant, ERα‐36, as well as GPER1 is present in embryonic stem cells. In addition, these receptors typically decrease their nuclear localisation as neuronal maturation proceeds. Finally, though these ERs are present in many subcellular compartments such as the nucleus and plasma membrane, we show that they are specifically not colocalised with each other, suggesting that they initiate distinct signalling pathways. This article is protected by copyright. All rights reserved.
... Following this treatment, NSC converted to flattened GFAP + cells similar to those observed in primary cultures of mouse astrocytes (Schildge et al., 2013; Figure 7A). In contrast, NOP differentiated into GFAP + cells displaying a mix of morphologies including flattened GFAP + cells, and cells with multiple thickened GFAP + processes typical of ESC derived astrocytes (Juneja et al., 2020; Figure 7B). Consistent with previous reports (Suzuki et al., 2017), we also detected GFAP + cells when cultures of pOPC were treated with BMP4 and LIF (data not shown). ...
... The NOP/OL population downregulates these NSC genes during differentiation in OL medium; thus, it would be interesting to compare their astroglial and neuronal potential with the more immature NOP cells. Regarding the identify of NOP-derived GFAP+ cells, BMP4 stimulation, as used here, is recognized to drive the differentiation of NPC toward an astroglial fate (Gross et al., 1996); thus, the GFAP+ cells, which had morphologies expected of cultured mouse astrocytes (Schildge et al., 2013;Juneja et al., 2020), are likely to represent astroglial lineage cells. Nevertheless, GFAP is also expressed by NPC/radial glia populations; thus, in the absence of other lineage markers, we term these cells astroglial-like. ...
Embryonic stem cells (ESC) have the potential to generate homogeneous immature cells like stem/progenitor cells, which appear to be difficult to isolate and expand from primary tissue samples. In this study, we developed a simple method to generate homogeneous immature oligodendrocyte (OL) lineage cells from mouse ESC-derived neural stem cell (NSC). NSC converted to NG2 ⁺ /OLIG2 ⁺ double positive progenitors (NOP) after culturing in serum-free media for a week. NOP expressed Prox1 , but not Gpr17 gene, highlighting their immature phenotype. Interestingly, FACS analysis revealed that NOP expressed proteins for NG2, but not PDGFRɑ, distinguishing them from primary OL progenitor cells (OPC). Nevertheless, NOP expressed various OL lineage marker genes including Cspg4 , Pdgfr α, Olig1/2 , and Sox9/10 , but not Plp1 genes, and, when cultured in OL differentiation conditions, initiated transcription of Gpr17 and Plp1 genes, and expression of PDGFRα proteins, implying that NOP converted into a matured OPC phenotype. Unexpectedly, NOP remained multipotential, being able to differentiate into neurons as well as astrocytes under appropriate conditions. Moreover, NOP-derived OPC myelinated axons with a lower efficiency when compared with primary OPC. Taken together, these data demonstrate that NOP are an intermediate progenitor cell distinguishable from both NSC and primary OPC. Based on this profile, NOP may be useful for modeling mechanisms influencing the earliest stages of oligogenesis, and exploring the cellular and molecular responses of the earliest OL progenitors to conditions that impair myelination in the developing nervous system.
... An abundance of neural differentiation protocols rely on the inclusion of growth factors in the medium. For example, RA and sonic hedgehog (SHH) or SHH agonists are commonly used in vitro to emulate the developmental differentiation pathway of motor neurons and astrocytes (Wichterle and Peljto, 2008;Hu and Zhang, 2009;Qu et al., 2014;Juneja et al., 2020). However, it is challenging to engineer complex structures (e.g., organoids) by using techniques that depend largely on the global application of chemical signals. ...
Alginate hydrogels are a commonly used substrate for in vitro 3D cell culture. These naturally derived biomaterials are highly tunable, biocompatible, and can be designed to mimic the elastic modulus of the adult brain at 1% w/v solution. Recent studies show that the molecular weight of the alginate can affect cell viability and differentiation. The relationship between the molecular weight, viscosity and ratio of G:M monomers of alginate hydrogels is complex, and the balance between these factors must be carefully considered when deciding on a suitable alginate hydrogel for stem cell research. This study investigates the formation of embryoid bodies (EB) from mouse embryonic stem cells, using low molecular weight (LMW) and high molecular weight (HMW) alginates. The cells are differentiated using a retinoic acid-based protocol, and the resulting aggregates are sectioned and stained for the presence of stem cells and the three germ layers (endoderm, mesoderm, and ectoderm). The results highlight that aggregates within LMW and HMW alginate are true EBs, as demonstrated by positive staining for markers of the three germ layers. Using tubular alginate scaffolds, formed with an adapted gradient maker protocol, we also propose a novel 3D platform for the patterned differentiation of mESCs, based on gradients of retinoic acid produced in situ by lateral motor column (LMC) motor neurons. The end product of our platform will be of great interest as it can be further developed into a powerful model of neural tube development.
